Alzheimer's disease (AD) is one of the most prevalent of progressive brain disorders. Currently an estimated 4.5 million older Americans suffer from AD. According to the latest estimates of the current and future prevalence of AD, the number of older people with AD will grow dramatically as the population ages. Projections indicate that as many as 13 million older Americans will have AD by 2050 unless new ways are found to prevent or treat the disease. AD is currently the third most expensive disease after heart disease and cancer. Treatment and care of those with AD now runs $100 to $150 billion a year and costs are projected to rise sharply as the population ages. Currently, pharmacological treatment of AD is primarily based on the use of acetylcholinesterase inhibitors (AChEIs), which have been reported to provide beneficial effects on cognitive, functional, and behavioral symptoms of the disease. Four of the five drugs approved for AD treatment in the U.S.—donepezil (Aricept®), rivastigmine (Exelon®), galantamine (Reminyl®), and tacrine (Cognex®)—are AChEIs. The fifth, Mementine (Namenda®), is an N-methyl-D-aspartate (“NMDA”) antagonist that alters glutamate signaling. Because only a small fraction of AD patients respond to this type of treatment, any new approach to the treatment or prevention of AD would have tremendous value. In spite of intensive research, there are no proven preventive agents or agents capable of limiting progression of the disease, and the few used to ameliorate its symptoms have side effects of nausea, vomiting, diarrhea, and even liver damage, yet do not meaningfully slow the disease's underlying course for most patients. AD may well be the most pharmaceutically under-served major disease in the US.
AD is physically evidenced by amyloid plaques and neurofibrillary tangles in the brain. These pathological markers are associated with cognitive regression and other varied symptoms of the disease. The amyloid plaques, which are the focus of the preponderance of research today on the disease, contain aggregated amyloid β-peptides derived from proteolytic cleavage of the larger amyloid precursor protein. The major component of neurofibrillary tangles is the protein tau, a constituent of the cytoskeleton. Tau is a microtubule-associated protein that functions in brain to regulate the structure and function of axonal microtubules. Over the past decade, several groups have demonstrated that the tau protein found in neurofibrillary tangles is hyperphosphorylated. Tau hyperphosphorylation is thought to destabilize microtubules and thereby contribute to neurodegenertion and the development of AD. Tau hyperphosphorylation results from an imbalance between kinase and phosphatase activities (reviewed in Buee et al., Brain Res Brain Res Rev 33(1): 95-130 (2000)). Several serine/threonine protein kinases have been implicated in tau phosphorylation including cyclin-dependent kinase 5 (“cdk-5”), glycogen synthase kinase-3b (“GSK-3b”) and MAP kinases. Tau dephosphorylation appears to be primarily mediated by protein phosphatase 2A (“PP2A”). Importantly, recent results suggest that a decrease in PP2A activity is associated with the elevated levels of tau phosphorylation that appear to cause neurofibrillary tangle formation (Planel et al., J Biol Chem. 276(36):34298-306 (2001)). Enhancement of this activity therefore may have significant therapeutic value. Since PP2A methylation greatly enhances the formation of a PP2A heterotrimer, it is believed that enhancing PP2A methylation will result in enhanced PP2A activity towards Tau.
Protein phosphatase 2A (PP2A) associates with a variety of regulatory subunits. (Janssens, V., Gloris, J., Biochem. J. 353 (Pt. 3): 417-39 (2001)). The predominant form in neuronal tissue is a trimer composed of a dimeric core composed of a 65 kilodalton (kDa) A subunit and the 36 kDa PP2A catalytic C subunit associated with one of several different regulatory B subunits. Whereas the A and C subunits are present more or less uniformly, the B subunit is variable and confers substrate specificity and subcellular localization to each PP2A holoenzyme trimer. The number and types of B subunits present is subject to developmental regulation and is cell type specific.
The variable B subunits of PP2A are classified into four families: (1) the B family with four isoforms (α, β, γ, δ); (2) the B′ family with five isoforms (α, β, γ, δ, ε); (3) the B″ family; and (4) the B′″ family. The PP2A ABαC heterotrimer appears to be the major phosphatase in brain responsible for dephosphorylation of tau. (Kamibayashi, C. et al., J. Biol. Chem. 269 (31): 20139-148 (1994); Sontag, E., et al., J. Neuropathol. Exp. Neurol. 63 (4): 287-301 (2004)).
The alpha-carboxyl of the C terminal leucine residue of the catalytic subunit of PP2A is subject to methyl esterification and methyl-ester hydrolysis, and the methylation state of PP2A regulates heterotrimer formation. (Tokstykh, T. et al., EMBO J. 19 (21): 5682-91 (2000); Wu, J. et al., EMBO J. 19 (21): 5672-81 (2000); Wei, H. et al., J. Biol. Chem. 276 (2): 1570-77 (2001); Yu, X X, et al., Mol. Biol. Cell 12 (1): 185-99 (2001)). Two enzymes are involved in controlling the methylation state of PP2A: (1) an S-adenosylmethionine-dependent PP2A-specific protein methyltransferase (“PPMT”), which adds the methyl group and (2) a PP2A-specific protein methylesterase (“PPME”), which removes the methyl group. PP2A methylation promotes PP2A ABαC trimer assembly. Any deficiency in methylation is expected to preclude PP2A ABαC heterotrimer formation, thereby leading to a deficiency in tau dephosphorylation, tau hyperphosphorylation and the formation of neurofibrillary tangles. (Vafai, S. B., Stock, J. B., FEBS Lett. 518 (1-3): 1-4 (2002)).
Homocysteine, a sulfur-containing amino acid that can be either remethylated to methionine or undergo a trans-sulfuration reaction to cystathionine, plays a key role in methylation metabolism (see FIG. 1). The conversion of homocysteine to methionine occurs in all tissues. Methionine is activated by ATP in the presence of methionine adenosyl transferase (labeled as (1) in FIG. 1) to form the methyl donor, S-adenosylmethionine (“SAM”). SAM-dependent methylation reactions in the presence of SAM-dependent methyltransferases (labeled as (2) in FIG. 1) result in the formation of S-adenosylhomocysteine (“SAH”), which is cleaved by SAH hydrolase (labeled as (3) in FIG. 1) to form adenosine and homocysteine. This reaction is reversible with the equilibrium favoring the condensation of homocysteine and adenosine. Under normal conditions, homocysteine is rapidly methylated, which favors the further cleavage of SAH. Homocysteine accumulation leads to global decreases in cellular methylation by the condensation of homocysteine with adenosine to form SAH, which acts as a product inhibitor in cellular methylation reactions. In the United States, 5-10% of the general population has elevated plasma homocysteine, and this imbalance increases to 30-40% in of the elderly. (Selub J., et al., Ann. Intern. Med. 131 (5): 331-39 (1999)). The enzymes cystathionine L-synthase; cystathionine Q lyase; betaine homocysteine methyltransferase; and methionine synthase are labeled as (4), (5), (6) and (7), respectively in FIG. 1. See Vafai, S. B., Stock, J. B., FEBS Lett. 2: 518 (2002).
Over the last several years, data has emerged in clinical literature suggesting a direct association between elevated plasma homocysteine and the occurrence of AD. Seshadri et al., (N Engl J Med 346 (7): 476-83 (2002)), demonstrated that elevated homocysteine is a risk factor for AD. After adjusting for other AD risk factors, the study concluded that plasma homocysteine levels greater than 14 μM coincided with about a 2-fold increased risk for developing AD with an additional 40% increased risk with each 5 μM incremental rise. Other diseases, conditions or disorders associated with elevated plasma homocysteine include, but are not limited to, atherosclerosis; neurodegenerative disorders, such as Parkinson's disease; cerebrovascular disorders (i.e., disorders pertaining to blood vessels in the brain), such as stroke; neuropsychiatric disorders, such as bipolar disorder and schizophrenia; diabetes (type II), and arthritis.
An analysis of the clinical and basic science literature indicates that a methylation defect resulting from elevated homocysteine could lead to lowered levels of PP2A methylation that would result in lowered PP2A ABαC, which is believed to lead to tau hyperphosphorylation, neurofibrillary tangle formation, and dementia (Vafai and Stock, FEBS Lett 518(1-3): 1-4 (2002)).
Cellular pathways for removing plasma homocysteine require folate, Vitamin B6 and B12, and thus high homocysteine levels are expected in mice fed diets deficient in these components. This was demonstrated using, male C57BL/J6 mice. One set of 4 week old mice were placed on a diet that contained folate, vitamin B6, and vitamin B12 and another set were fed diets that lacked these vitamins. The mice were allowed free access to both food and water. After nine weeks on their respective diets, each mouse was sacrificed by cervical dislocation. Blood samples were collected for measurement of plasma homocysteine and the brain was removed and quickly frozen in liquid nitrogen for further analysis of tau phosphorylation. As expected the vitamin-deficient diets caused substantial increases in plasma Hcy and brain SAH. These increases were accompanied by elevated levels of Tau phosphorylation, as shown in FIG. 2 FIG. 2 provides Western blots after SDS-PAGE of extracts prepared from the brains of mice raised on normal diets (A and B) and vitamin-deficient diets (C and D). CP13 and PHF1 are monoclonal antibodies that are specific for phosphorylated tau epitopes. TG5 is a monoclonal antibody that recognizes tau independent of its state of phosphorylation; it thereby provides a control showing that total levels of tau expression are unaffected by diet. Mice raised on diets deficient in folate, B12, and B6 had dramatically elevated levels of total plasma homocysteine, brain S-adenosyl homocysteine and elevated levels of tau phosphorylation. S-Adenosyl methionine levels were not significantly affected.
The demographics of aging in the United States population, combined with a lack of effective treatments, have heightened the need for AD therapies. Moreover, the development of preventives would be an even greater contribution to public health. A protective agent that could be taken over many years to reduce the risk of AD or to substantively delay its onset would be an invaluable breakthrough.
Coffee has been used for centuries by a diverse range of populations and is presently the most popular beverage worldwide with over 400 billion cups consumed each year. There are many anecdotal reports of the medicinal value of coffee but in spite of its worldwide prevalence, little is really known about its potential medical uses. Some epidemiological studies have suggested an inverse association between coffee consumption and the risk not only of AD, but also of liver cirrhosis, colorectal cancer, cardiovascular mortality, type 2 diabetes and Parkinson's disease. Recent studies have suggested that coffee consumption reduces the risk for AD by as much as 30% (Lindsay et al., Am J Epidemiol. 156(5):445-53 (2002)). Various mechanisms for the purported benefits have been suggested, but none have been explored fully enough for these suggestions to be definitive. Moreover, brewed coffee is a complex mixture that contains several pharmacologically active components, including caffeine.
The present invention provides a definitive mechanism, i.e., enhancement of PP2A methylation, by which to measure benefit to a subject's health and general well-being, and, in particular, cognitive function. An agent that increases the levels of PP2A methylation would help maintain high levels of PP2A ABC heterotrimer formation and may thereby prevent the demethylation of PP2A and the lowered levels of PP2A ABC heterotrimers that have been associated with tau hyperphosphorylation, neurofibrillary tangle formation, and neurodegeneration in AD.